A Reduced Risk of Infection with Plasmodium vivax and Clinical Protection against Malaria Are Associated with Antibodies against the N Termi
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感染与免疫杂志 2006年第5期
Instituto de Pesquisa em Patologias Tropicais, Estrada BR 364-Km 41, Porto Velho, Rondnia 78900-000
Departamento de Parasitologia, Instituto de Ciências Biomedicas, Universidade de So Paulo, Av. Lineu Prestes 1374, So Paulo SP 05508-900, Brazil
ABSTRACT
Progress towards the development of a malaria vaccine against Plasmodium vivax, the most widely distributed human malaria parasite, will require a better understanding of the immune responses that confer clinical protection to patients in regions where malaria is endemic. The occurrence of clinical protection in P. vivax malaria in Brazil was first reported among residents of the riverine community of Portuchuelo, in Rondnia, western Amazon. We thus analyzed immune sera from this same human population to determine if naturally acquired humoral immune responses against the merozoite surface protein 1 of P. vivax, PvMSP1, could be associated with reduced risk of infection and/or clinical protection. Our results demonstrated that this association could be established with anti-PvMSP1 antibodies predominantly of the immunoglobulin G3 subclass directed against the N terminus but not against the C terminus, in spite of the latter being more immunogenic and capable of natural boosting. This is the first report of a prospective study of P. vivax malaria demonstrating an association of reduced risk of infection and clinical protection with antibodies against an antigen of this parasite.
INTRODUCTION
Malaria remains one of the major public health problems in South and Central America, where Plasmodium vivax is the main species responsible for infections. Although malaria caused by this human parasite is not lethal, the clinical manifestations represent a major socioeconomical burden in the major cities and rural areas of the Brazilian Amazon, which contains 99.7% of all malaria cases. Although a few studies indicate that some cross-protection between Plasmodium vivax, the most widely distributed human malaria parasite, and P. falciparum, the most virulent species, does occur (33), immunity in malaria is mostly species specific; thus, it is likely that a vaccine against P. falciparum will not cross-protect against P. vivax. Progress is being made to develop P. vivax vaccines per se (for a review, see reference 2); however, to date there are no prospective studies associating human immune responses to any P. vivax antigen with clinical protection.
The history of malaria in Brazil has been punctuated with epidemics associated with migration movements of nonimmune population to areas where malaria is endemic (20, 30, 36). Previous studies done in these epidemiological settings showed that Plasmodium infection was always associated with symptoms, and clinical protection was not observed (27). However, through a cross-sectional and longitudinal study among native Amazon residents of the riverine community of Portuchuelo in Rondnia, located in the western Brazilian Amazon, the occurrence of symptomless Plasmodium vivax-infected individuals was reported (1, 6). Symptomless Plasmodium infections were defined as individuals who contain parasites in their peripheral blood, as detected by Giemsa blood smears and/or PCR, and who were not drug treated yet did not develop clinical symptoms during a 2-month individual follow-up. These rigorous criteria underpin the importance of these patients from which immune sera, full clinical, parasitological, and epidemiological data are available and who can unequivocally be considered the first human population in which clinical protection has been described and documented for P. vivax in Brazil.
Naturally acquired immunoglobulin G (IgG) antibodies against merozoite surface antigens of Plasmodium play a major role in acquired immunity to malaria. Among these antigens, the merozoite surface protein 1 (MSP1) has received the most attention, as it is presently considered a leading vaccine candidate against the asexual blood stages of P. falciparum and P. vivax (for reviews, see references 2 and 18). MSP1 is a large-molecular-mass protein synthesized as a precursor and later processed into four major fragments of circa 83 kDa, 30 kDa, 38 kDa, and 42 kDa. A second specific processing step cleaves the 42-kDa C-terminal fragment into a 33-kDa polypeptide that is shed into circulation and a 19-kDa portion (MSP119) that remains attached to the newly formed ring stage parasite after invasion (16). Antibodies against different regions of MSP1 of P. falciparum (PfMSP1) are immunogenic in natural infections and are associated with reduced clinical symptoms. Thus, early studies have demonstrated the presence of naturally acquired antibodies against the N terminus (7, 9) and C terminus (13, 28, 31) of PfMSP1. The presence of such naturally acquired antibodies against MSP1 was later correlated to clinical protection in some, though not all, studies (8, 10, 12, 14, 24, 25, 26). Although the role of other portions of this large molecule remains mostly unknown, these studies have validated the N and C termini of PfMSP1 as solid subunit vaccine candidates against P. falciparum.
Studies on naturally acquired IgG responses against the MSP1 protein of Plasmodium vivax (PvMSP1) were initiated after the primary structure of the gene encoding this antigen revealed the existence of conserved and polymorphic blocks among different Plasmodium species (11). Thus, recombinant proteins representing conserved and polymorphic regions from the N terminus of PvMSP1 demonstrated that polymorphic, as opposed to conserved regions, of PvMSP1 are immunogenic in natural infections and that close to 50% of patients with multiple infections had an antibody response in which the predominant isotype was IgM (19, 22). Studies including the C terminus of PvMSP1 followed and demonstrated that this region is the most immunogenic portion of the molecule, that the presence of antibodies against it is associated with recent malaria attacks, and that it is the only region of PvMSP1 capable of boosting upon new infections (34, 35). To date, however, no association of clinical protection and/or reduced risk of infection in P. vivax and PvMSP1 or any other parasite antigen has been reported. The aim of this study was to determine if the presence of naturally acquired IgG antibodies against the N terminus and/or C terminus of PvMSP1 was associated with reduced risk of infection and/or clinical protection against P. vivax in individuals from the human population of Portuchuelo.
MATERIALS AND METHODS
Human population and area. Riverine communities in the Amazon are constituted by descendants of local aboriginal populations and migrants from northeast Brazil (from the late 19th century to the early 20th century) who live on subsistence fishing and farming. The studied human population consisted of 180 individuals living in the riverine community of Portuchuelo, settled on the riverbanks of Rio Madeira in the western Brazilian Amazon state of Rondnia (Fig. S1 in the supplemental material). The majority of individuals (92%) from Portuchuelo community were born in the Amazon region and were exposed to malaria since birth. Because of geographical and malaria epidemiological differences, the Portuchuelo community was divided into Sector A, which was more accessible and had lower malaria infection risk than Sector B, a more remote area only reachable by boat (17) (Fig. S1). Malaria is seasonal in this region, peaking at the end of the rainy season, in March-April. Clinical, parasitological, and epidemiological data from this human population were recorded through cross-sectional surveys and a prospective longitudinal study in 1998 to 1999 (1).
Sample collection. During each cross-sectional survey (survey A, September 1998; B, March 1999; C, September 1999), the villagers were interviewed and examined. Upon informed consent from the patients, 5 ml of peripheral blood was collected by venipuncture, and blood was processed the same day at the laboratory of the Instituto de Apoio a Pesquisas em Patologias Tropicais in Porto Velho. Sera were stored at –20°C in 50% glycerol, and Plasmodium spp. infections were diagnosed by microscopy (Giemsa staining) and nested PCR (32). These studies received ethical clearance from the local ethics committee and the University of So Paulo Ethics Committee.
Recombinant proteins. Glutathione S-transferase (GST) fusion proteins representing the N terminus (ICB2-5; contains 505 amino acids [aa], corresponding to aa 170 to 675 from the original PvMSP1 molecule of the Belem strain [11]) and the C terminus (ICB10; contains 111 amino acids, corresponding to aa 1615 to 1726, and encodes the two epidermal growth factor-like domains of other MSP119 molecules) of PvMSP1 have already been described (19, 34). GST and recombinant proteins were purified on glutathione-Sepharose 4B columns (Amersham Pharmacia), and protein concentration was determined by Bradford assay (Bio-Rad).
Immunoassay (enzyme-linked immunosorbent assay [ELISA]). The presence of naturally acquired antibodies against GST-PvMSP1 tags has been described in detail elsewhere (19, 34). To determine if a serum was positive, we calculated the cutoff for each recombinant protein representing the N terminus and the C terminus of PvMSP1 for each survey, using as negative controls individual sera from nine healthy individuals from Portuchuelo and three healthy individuals from elsewhere who never had a past history of malaria. The cutoff was calculated by the average of ICB2-5 optical density (OD) minus GST OD plus 2 standard deviations (SD). All tests were done in duplicate. Average OD was calculated for each individual, and serum was considered positive if ICB2-5 OD minus GST OD was equal to or greater than the cutoff. The same methodology was applied for ICB10.
IgG subclasses. IgG subclasses were determined by ELISA as described elsewhere (5). Briefly, mouse monoclonal antibodies to each subclass were purchased from Sigma (St. Louis, MO) and diluted according to manufacturer's instructions using a panel of positive sera previously known to react with each subclass. All sera were tested at 1:100 dilutions, and monoclonal antibody binding was detected with peroxidase-conjugated anti-mouse immunoglobulin (Sigma). Positive sera for each isotype were determined as described above for total IgG.
Statistical analyses. Microsoft Access 2.0 (Redmond, WA) was used for database storage. Analysis was performed with SPSS version 10.0 and Epi Info 3.3.2 (Centers for Disease Control and Prevention, Atlanta, GA). Proportions and categorical data were compared by chi-squared test with Yate's correction in cases of 2-by-2 contingency tables or by Fisher's exact test. Odds ratios (OR) and 95% confidence intervals (95% CI) were used to measure associations. Continuous variables analyzed in this data set did not conform to normal distribution and thus were analyzed by Mann-Whitney tests. Multivariate logistic regression analysis was used to evaluate (i) the association between PvMSP1 serology and time of residence in areas were malaria is endemic, controlling for age, and (ii) the association between malaria clinical outcome (symptomatic versus asymptomatic infection) and serology against the PvMSP1 N terminus and C terminus in survey A, controlling for age. Kaplan-Meier survival analysis was performed to compare the probability of P. vivax infection over the 1-year follow-up period (September 1998 to September 1999) in the groups of individuals with positive and negative serology against PvMSP1 N and C termini in survey A. Because malaria is seasonal in Portuchuelo (Fig. 1), only individuals who remained in Portuchuelo the entire study period were included in the survival analysis. Moreover, individuals who were found infected in survey A were excluded due to the uncertainty of their serology against PvMSP1 at the time they were infected. Because individuals living in Sector B (Fig. S1) of Portuchuelo are at higher risk of malaria infection (17), a Cox proportional hazard model was further used to evaluate the risk of P. vivax infection by serology, controlling for geographical sector.
RESULTS
Prevalence of antibodies against PvMSP1 in the study area. The prevalence of anti-PvMSP1 antibodies was determined using immune sera collected from individuals living in Portuchuelo in three different cross-sectional surveys: survey A, September 1998 (n = 173); survey B, March 1999 (n = 125); and survey C, September 1999 (n = 131). Moreover, as both the N and C termini of PvMSP1 are immunogenic in natural infections (19, 34), we determined the prevalence of anti-PvMSP1 antibodies against each of these regions using GST-recombinant proteins representing the N terminus and the C terminus in ELISA assays. It is notable that during the year of the study, the incidence of P. vivax infections per 1,000 inhabitants was seasonal, peaking at the end of the rainy season when the second survey (B) was performed (Fig. 1). In spite of these marked differences in incidences during the study year, the prevalence of anti-PvMSP1 antibodies against the N and C termini of PvMSP1 was not significantly different in all three surveys (N terminus for survey A, 36.4%; N terminus for survey B, 32.8%; N terminus for survey C, 35.1%; C terminus for survey A, 42.8%; C terminus for survey B, 42.4%; C terminus for survey C, 38.9%). Moreover, although the overall prevalence of antibodies against the PvMSP1 C terminus in each survey was higher than that of antibodies against the PvMSP1 N terminus, these differences were not statistically significant (A, P = 0.23; B, P = 0.12; and C, P = 0.52). It was also observed that the presence of antibodies to the N terminus was associated with the presence of antibodies to the C terminus of PvMSP1 in surveys A and B (P = 0.002 for both), while in survey C, although the same trend was observed, the difference was not statistically significant (P = 0.085). These results showed that the N and C termini of PvMSP1 are immunogenic in natural infections among the residents of Portuchuelo and validated their use as markers for looking for associations with reduced risk of infection and clinical protection.
Dependency of antibody responses to PvMSP1 and time of residence in areas where malaria is endemic, age, and time since last malaria attack. To determine whether the antibody responses against PvMSP1 were dependent on the time of residence in areas where malaria is endemic, we grouped time of residence as 0 to 7, 8 to 14, 15 to 29, and more than 30 years based on 25th percentile of all three cross-sectional surveys (Tables 1 and 2). A multiple logistic regression analysis was performed to evaluate if serology against PvMSP1 was associated with time living in areas where malaria is endemic, controlling for age. Odds ratios (with 95% CI) were calculated for each group, using 0 to 7 years as the reference group. The results demonstrated that individuals living longer in areas where malaria is endemic had increasingly higher chances of having naturally acquired IgG antibodies against the N terminus but not against the C terminus of PvMSP1. Thus, for the N terminus there was no statistically significant difference between the reference group and the group of 8 to 14 years. However, individuals living in areas where malaria is endemic for 15 to 29 years or for 30 or more years had 30 times greater chances of having acquired antibodies against the PvMSP1 N terminus (Table 1). Similar results were observed in surveys B and C, although for survey C it was not possible to run the logistic regression and calculate the odds ratio, because no individuals in the reference group had positive serology (but the same trend was clear; 2 = 67.5, P < 0.001). In contrast, the same analysis for the C terminus of PvMSP1 showed no statistically significant differences among the groups in the three surveys (Table 2). These results demonstrate that acquisition of antibodies to the N terminus but not the C terminus of PvMSP1 in residents of Portuchuelo depends on the length of residence in areas where malaria is endemic.
We next analyzed if there was a dependency in the acquisition of antibodies against the N and C termini of PvMSP1 with age and the amount of time since the last malaria attack. As these parameters did not present a normal distribution for this population, we used the nonparametric test of Mann-Whitney for analysis. As expected, the results with regard to age demonstrated that individuals with positive serology against the N terminus of PvMSP1 are older than individuals with negative serology for all three surveys (survey A, median = 38 versus 13 years, Mann-Whitney = 1,468, P < 0.0001; survey B, median = 25 versus 13 years, Mann-Whitney = 955.5, P < 0.0001; survey C, median = 37 versus 11 years, Mann-Whitney = 582.5, P < 0.0001). Similar results were observed for the PvMSP1 C terminus only in survey A (median = 20.5 versus 16 years, Mann-Whitney = 2,981, P = 0.036). For surveys B and C, the difference was not significant (survey B, median = 21 versus 15.5 years, Mann-Whitney = 1,540, P = 0.07; survey C, median = 15 versus 13 years, Mann-Whitney = 1,617, P = 0.06). This result is likely due to the fact that the risk of infection is the same for all age groups in Portuchuelo (1), and there is a natural boost against the PvMSP1 C terminus but not the N terminus (34). Thus, upon infection during survey B, many different individuals reacted and remained positive against the PvMSP1 C terminus independently of age.
Analysis of the dependency of anti-PvMSP1 antibodies on the amount of time since the last malaria attack revealed that individuals with positive serology against the PvMSP1 N terminus had their most recent malaria attack significantly farther in the past than those with negative serology (survey A, median = 6 versus 2 years, Mann-Whitney = 1,449, P < 0.0001). In contrast, there were no significant differences for individuals with positive and negative serology against the PvMSP1 C terminus and the amount of time since the last malaria attack (survey A, median = 2 versus 3 years, Mann-Whitney = 2,698, P = 0.61). Similar results for both PvMSP1 termini were observed in surveys B and C (data not shown). Together, these results showed a dependency on the acquisition of IgG antibodies against the N terminus of PvMSP1 and time of residence in areas where malaria is endemic, age, and time since the last malaria attack. The same dependency was not observed for the C terminus of PvMSP1. As time of residence in areas where malaria is endemic had been associated with acquired immunity in the epidemiological study of the same human population (1), we next determined if an association could be established with PvMSP1, risk of infection, and clinical protection.
Association of antibody responses to PvMSP1 with risk of P. vivax infection and clinical protection. To compare the probability of P. vivax infection in the groups of individuals with positive and negative responses against PvMSP1 in survey A, a survival analysis of time to P. vivax infection over the 1-year follow-up period was performed. The Kaplan-Meier cumulative risk of P. vivax infection at the end of the study year was significantly lower for individuals with positive serology in survey A than for individuals with negative serology against the PvMSP1 N terminus (11.9% versus 31.8%, respectively; P = 0.0186). In contrast, this association was not significant for individuals with positive or negative serology against the PvMSP1 C terminus (26.9% versus 23.5%, respectively; P = 0.7414) (Fig. 2). Because individuals living in Sector B of Portuchuelo are at higher risk of malaria infection (17), a Cox proportional hazard model was further used to evaluate the relative risk of P. vivax infection for the groups of individuals with positive and negative serology against PvMSP1, controlling for geographical sector. Indeed, the risk of P. vivax infection after adjusting for sector was three times higher for individuals with negative serology than for individuals with positive serology against the N terminus (hazard ratio, 2.954; 95% CI, 1.140 to 7.652; P = 0.026) but not the C terminus (hazard ratio, 0.892; 95% CI, 0.447 to 1.778; P = 0.745) of PvMSP1.
Having established that individuals with positive serology against the PvMSP1 N terminus had reduced risk of P. vivax infection, we next performed a multivariate logistic regression analysis to determine if malaria clinical outcome (symptomatic versus asymptomatic) was associated with positive serology against PvMSP1 in survey A, controlling for age. As shown in Table 3, individuals with positive serology against the N terminus had chances of having asymptomatic P. vivax infection, after adjusting for age, that were 12 times greater than those of individuals with positive serology against the PvMSP1 C terminus. Together, these data show that individuals with positive serology against the PvMSP1 N terminus but not the C terminus are protected against P. vivax infection, and when they are infected, they are likely to be symptomless.
IgG subclass response to PvMSP1 N and C termini. To determine if there was a particular IgG subclass correlated with protection, we examined the IgG isotype responses of patients presented in the three surveys who had a demonstrated presence of PvMSP1 total IgG. No IgG2 or IgG4 response to either the N or C terminus of PvMSP1 was detected (data not shown). In contrast, there were marked differences in the frequencies of IgG1 and IgG3 isotype responses in all three surveys to the N and C termini (Fig. 3). Thus, IgG1 isotype response was of low frequency or was nondetectable in all three surveys for either region of PvMSP1 (Fig. 3). In striking contrast, high frequencies of IgG3 isotype response were detected to the N terminus (56% in survey A, 63.2% in survey B, and 69.6% in survey C) but not to the C terminus (0% in survey A, 16% in survey B, and 3.6% in survey C) of PvMSP1. Indeed, the frequency of IgG3 response was significantly higher for the N terminus than the C terminus for all three surveys (P < 0.003).
Longitudinal analysis of anti-PvMSP1 antibodies in asymptomatic versus symptomatic P. vivax individuals from Portuchuelo. We next examined the relative levels of anti-PvMSP1 antibodies in asymptomatic and symptomatic P. vivax individuals from Portuchuelo in the three cross-sectional surveys. To do so, we recorded individual ELISA OD minus GST OD values of the sera against the recombinant proteins representing the N terminus and C terminus of PvMSP1. Responses were classified as negative (OD < cutoff) or positive; positives were subgrouped according to the 33rd and 66th percentiles as low (cutoff < OD < 33rd percentile), middle (33rd percentile < OD < 66th percentile), and high (OD p66th percentile). As shown in Fig. S2 in the supplemental material, asymptomatic individuals present a higher proportion and magnitude of response to the N terminus of PvMSP1 than do symptomatic individuals, while this difference is not observed for the C terminus of PvMSP1.
DISCUSSION
In this work, a prospective 1-year longitudinal study of a human population in the Brazilian Amazon Basin in which asymptomatic P. vivax patients had been previously identified was performed to determine if the presence of naturally acquired antibodies against the N terminus and/or C terminus of the merozoite surface protein 1 of Plasmodium vivax (PvMSP1) was associated with reduced risk of P. vivax infection and clinical protection. The results demonstrated that close to 33% and 40% of the population contained anti-PvMSP1 antibodies against the N and C termini of PvMSP1, respectively, independent of seasonal transmission rates. Moreover, the data showed that individuals living in areas where malaria is endemic for 15 to 29 years or 30 years had 30 times greater chances of having acquired antibodies against the PvMSP1 N terminus, but not the C terminus, than individuals living <7 years in areas where malaria is endemic. Acquisition of antibodies against the N terminus but not the C terminus of PvMSP1 was dependent on age and the amount of time since the last malaria attack. Moreover, when an association of anti-PvMSP1 antibodies with reduced risk of P. vivax infection and clinical protection was investigated in the individuals who participated in this study for the entire year, we found that this association could only be established with antibodies against the N terminus but not the C terminus of PvMSP1 and that most asymptomatic individuals presented IgG antibodies predominantly of the immunoglobulin G3 subclass against the N terminus of PvMSP1.
The existence of asymptomatic plasmodial infections in Brazil allowed the initiation of studies to look for associations of antibodies against parasite antigens with clinical protection (5, 23). Interestingly, these two reports used the same human populations to look for associations of acquired immunity with IgG subclasses against the C terminus of the MSP1 proteins from P. falciparum (5) and P. vivax (23). The criteria used to define asymptomatic individuals in these studies (Giemsa-positive or PCR-positive patients that did not develop symptoms after 72 h of this detection), however, is not stringent enough to discount the possibility that these could later turn into symptomatic patients. Regardless, the results suggested that there is an association of clinical protection with IgG1 subclass antibodies against the C terminus of P. falciparum (5), whereas no association with regard to antibodies or subclasses could be supported against the C terminus of P. vivax (23).
In contrast to the above studies, we looked for associations of clinical protection and reduced risk of infection with serology against PvMSP1 in a human population in which symptomless P. vivax-infected individuals have been unarguably found. In fact, one of us (F.A.P.) personally diagnosed these patients, and after obtaining ethical consent to not treat them with drugs, actively followed them up twice a week for 2 months to guarantee that they did not develop malaria symptoms. Moreover, we analyzed independently the N and C termini of PvMSP1, as antibodies against both regions of the MSP1 of P. falciparum have been associated with clinical protection and acquired immunity (8, 10, 12, 14, 24, 25, 26). Strikingly, in spite of being the most immunogenic portion of PvMSP1 in natural infections and being capable of boosting (34), there was no association of antibody responses against the C terminus of PvMSP1 in survey A with clinical protection or risk of P. vivax infection (Fig. 2 and Table 3). This result is thus in agreement with the one reported previously for this portion of PvMSP1 (23). In contrast, immune sera from these same patients revealed a highly statistically significant association of antibodies against the N terminus of PvMSP1 with clinical protection and reduced risk of P. vivax infection over the 1-year follow-up period. Prospective studies with other human populations in which symptomless P. vivax-infected individuals are solidly identified should validate whether this finding is a particularity of individuals from Portuchuelo or a general finding for vivax malaria in Brazil and elsewhere.
It is now amply recognized that clinical protection against the asexual blood stages in falciparum malaria is associated with the cytophilic IgG1 and IgG3 subclass antibodies directed against merozoite surface antigens (3, 4). Indeed, solid associations of clinical protection of IgG3 isotype antibodies against MSP2 in the Solomon Islands and in The Gambia (29, 37) and of the IgG1 isotype to RAP1 in reference 15 have been reported. In the particular case of the MSP1 protein of P. falciparum, initial studies using recombinant proteins representing the N and C termini of PfMSP1 clearly demonstrated that there is a marked dichotomy in the IgG subclass response to these regions (7). Thus, the IgG3 subclass response is predominantly against block 2 from the N terminus, whereas the IgG1 subclass response is predominantly against MSP119 at the C terminus. Our findings demonstrated that only the IgG3 subclass response is frequently detected in asymptomatic individuals, and this response is against the N terminus but not the C terminus of PvMSP1. Further work needs to be done to determine why merozoite surface antigens are able to elicit such strikingly different IgG subclass responses to different regions of the same molecules and the consequences of such responses with regard to clinical aspects of malaria.
An interesting observation of our results is that high sustained levels of antibodies against the N terminus of PvMSP1 were only detected in asymptomatic individuals, whereas in symptomatic patients, most of them did not recognize this recombinant protein (Fig S2). In comparison, similar levels of antibodies against the C terminus of PvMSP1 were seen in both asymptomatic and symptomatic patients. Sustained high antibody levels against the N terminus of PvMSP1 in asymptomatic patients can be explained by the presence of circulating, mostly undetectable parasites, which should facilitate the formation and migration of specific plasma cells to the bone marrow (21). Lack of high levels of antibodies against the N terminus of PvMSP1 in symptomatic patients is far more difficult to explain, but it seems clear that an active mechanism precludes most of these patients from developing an antibody response against this region of PvMSP1. Whether this observation and mechanism are indeed related to acquired immunity in P. vivax remains to be determined.
The existence of asymptomatic P. vivax-infected individuals poses a major problem for eradication control strategies in regions where malaria is endemic, as they represent reservoirs that maintain circulating parasites. Giemsa staining and PCR first helped in the identification of such asymptomatic carriers. Our studies point to the potential value of the PvMSP1 antibodies, in particular of the IgG3 subclass against the N terminus of PvMSP1, as seroepidemiological markers that, together with information on time of residence in areas where malaria is endemic, can indicate the existence of clinically protected individuals in a population. Active surveillance of human populations using these three techniques (microscopy, PCR, and serology) together with epidemiological data should unveil most asymptomatic carriers and guide local health policies to treat them with drugs as an additional malaria control strategy. It has been shown that such a procedure significantly reduces malaria cases (F. P. Alves, L. H. Pereira da Silva, and E. P. Camargo, unpublished data).
In summary, our study is the first to demonstrate an association of clinical protection and reduced risk of infection with naturally acquired IgG antibodies, predominantly of the IgG3 subclass, against a P. vivax antigen, PvMSP1. This association was with the N terminus but not the C terminus of PvMSP1, in spite of the latter being the most immunogenic portion of the molecule and the only portion capable of boosting in natural infections. It is important to emphasize, however, that this association does not imply that antibody responses to the PvMSP1 N terminus is by itself the mechanism of protection or simply a marker of it. Regardless, fine epitope mapping of N-terminus PvMSP1 sequences associated with this clinical protection and reduced risk of infection should guide rational approaches to develop a subunit PvMSP1 N-terminus vaccine.
ACKNOWLEDGMENTS
This work was supported by the Fundao de Amparo a Pesquisa do Estado de So Paulo (FAPESP), Coordenao de Aperfeioamento de Pessoal de Nível Superior (CAPES), Programa de Apoio a Núcleos de Excelência (PRONEX) do Ministerio da Ciência e Tecnologia, Brasil, and Program PAL+ (Recherche sur le paludisme et les maladies transmissibles associees pour les pays em development), Institut de Recherche pour le Development, France. The laboratory of H.A.P. receives support from FAPESP (01/09401-0) and CNPq (302572/2002-3).
We are particularly grateful to Irene Soares for titrations and helpful discussions on malarial MSP1 IgG subclasses and to Andrea Sendoda and Ricardo Raele for helping in purifying recombinant proteins and aliquoting the sera in 96-well plates.
Supplemental material for this article may be found at http://iai.asm.org/.
These authors contributed equally to this work.
Present address: DKFZ INF 280, 69120 Heidelberg, Germany.
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Departamento de Parasitologia, Instituto de Ciências Biomedicas, Universidade de So Paulo, Av. Lineu Prestes 1374, So Paulo SP 05508-900, Brazil
ABSTRACT
Progress towards the development of a malaria vaccine against Plasmodium vivax, the most widely distributed human malaria parasite, will require a better understanding of the immune responses that confer clinical protection to patients in regions where malaria is endemic. The occurrence of clinical protection in P. vivax malaria in Brazil was first reported among residents of the riverine community of Portuchuelo, in Rondnia, western Amazon. We thus analyzed immune sera from this same human population to determine if naturally acquired humoral immune responses against the merozoite surface protein 1 of P. vivax, PvMSP1, could be associated with reduced risk of infection and/or clinical protection. Our results demonstrated that this association could be established with anti-PvMSP1 antibodies predominantly of the immunoglobulin G3 subclass directed against the N terminus but not against the C terminus, in spite of the latter being more immunogenic and capable of natural boosting. This is the first report of a prospective study of P. vivax malaria demonstrating an association of reduced risk of infection and clinical protection with antibodies against an antigen of this parasite.
INTRODUCTION
Malaria remains one of the major public health problems in South and Central America, where Plasmodium vivax is the main species responsible for infections. Although malaria caused by this human parasite is not lethal, the clinical manifestations represent a major socioeconomical burden in the major cities and rural areas of the Brazilian Amazon, which contains 99.7% of all malaria cases. Although a few studies indicate that some cross-protection between Plasmodium vivax, the most widely distributed human malaria parasite, and P. falciparum, the most virulent species, does occur (33), immunity in malaria is mostly species specific; thus, it is likely that a vaccine against P. falciparum will not cross-protect against P. vivax. Progress is being made to develop P. vivax vaccines per se (for a review, see reference 2); however, to date there are no prospective studies associating human immune responses to any P. vivax antigen with clinical protection.
The history of malaria in Brazil has been punctuated with epidemics associated with migration movements of nonimmune population to areas where malaria is endemic (20, 30, 36). Previous studies done in these epidemiological settings showed that Plasmodium infection was always associated with symptoms, and clinical protection was not observed (27). However, through a cross-sectional and longitudinal study among native Amazon residents of the riverine community of Portuchuelo in Rondnia, located in the western Brazilian Amazon, the occurrence of symptomless Plasmodium vivax-infected individuals was reported (1, 6). Symptomless Plasmodium infections were defined as individuals who contain parasites in their peripheral blood, as detected by Giemsa blood smears and/or PCR, and who were not drug treated yet did not develop clinical symptoms during a 2-month individual follow-up. These rigorous criteria underpin the importance of these patients from which immune sera, full clinical, parasitological, and epidemiological data are available and who can unequivocally be considered the first human population in which clinical protection has been described and documented for P. vivax in Brazil.
Naturally acquired immunoglobulin G (IgG) antibodies against merozoite surface antigens of Plasmodium play a major role in acquired immunity to malaria. Among these antigens, the merozoite surface protein 1 (MSP1) has received the most attention, as it is presently considered a leading vaccine candidate against the asexual blood stages of P. falciparum and P. vivax (for reviews, see references 2 and 18). MSP1 is a large-molecular-mass protein synthesized as a precursor and later processed into four major fragments of circa 83 kDa, 30 kDa, 38 kDa, and 42 kDa. A second specific processing step cleaves the 42-kDa C-terminal fragment into a 33-kDa polypeptide that is shed into circulation and a 19-kDa portion (MSP119) that remains attached to the newly formed ring stage parasite after invasion (16). Antibodies against different regions of MSP1 of P. falciparum (PfMSP1) are immunogenic in natural infections and are associated with reduced clinical symptoms. Thus, early studies have demonstrated the presence of naturally acquired antibodies against the N terminus (7, 9) and C terminus (13, 28, 31) of PfMSP1. The presence of such naturally acquired antibodies against MSP1 was later correlated to clinical protection in some, though not all, studies (8, 10, 12, 14, 24, 25, 26). Although the role of other portions of this large molecule remains mostly unknown, these studies have validated the N and C termini of PfMSP1 as solid subunit vaccine candidates against P. falciparum.
Studies on naturally acquired IgG responses against the MSP1 protein of Plasmodium vivax (PvMSP1) were initiated after the primary structure of the gene encoding this antigen revealed the existence of conserved and polymorphic blocks among different Plasmodium species (11). Thus, recombinant proteins representing conserved and polymorphic regions from the N terminus of PvMSP1 demonstrated that polymorphic, as opposed to conserved regions, of PvMSP1 are immunogenic in natural infections and that close to 50% of patients with multiple infections had an antibody response in which the predominant isotype was IgM (19, 22). Studies including the C terminus of PvMSP1 followed and demonstrated that this region is the most immunogenic portion of the molecule, that the presence of antibodies against it is associated with recent malaria attacks, and that it is the only region of PvMSP1 capable of boosting upon new infections (34, 35). To date, however, no association of clinical protection and/or reduced risk of infection in P. vivax and PvMSP1 or any other parasite antigen has been reported. The aim of this study was to determine if the presence of naturally acquired IgG antibodies against the N terminus and/or C terminus of PvMSP1 was associated with reduced risk of infection and/or clinical protection against P. vivax in individuals from the human population of Portuchuelo.
MATERIALS AND METHODS
Human population and area. Riverine communities in the Amazon are constituted by descendants of local aboriginal populations and migrants from northeast Brazil (from the late 19th century to the early 20th century) who live on subsistence fishing and farming. The studied human population consisted of 180 individuals living in the riverine community of Portuchuelo, settled on the riverbanks of Rio Madeira in the western Brazilian Amazon state of Rondnia (Fig. S1 in the supplemental material). The majority of individuals (92%) from Portuchuelo community were born in the Amazon region and were exposed to malaria since birth. Because of geographical and malaria epidemiological differences, the Portuchuelo community was divided into Sector A, which was more accessible and had lower malaria infection risk than Sector B, a more remote area only reachable by boat (17) (Fig. S1). Malaria is seasonal in this region, peaking at the end of the rainy season, in March-April. Clinical, parasitological, and epidemiological data from this human population were recorded through cross-sectional surveys and a prospective longitudinal study in 1998 to 1999 (1).
Sample collection. During each cross-sectional survey (survey A, September 1998; B, March 1999; C, September 1999), the villagers were interviewed and examined. Upon informed consent from the patients, 5 ml of peripheral blood was collected by venipuncture, and blood was processed the same day at the laboratory of the Instituto de Apoio a Pesquisas em Patologias Tropicais in Porto Velho. Sera were stored at –20°C in 50% glycerol, and Plasmodium spp. infections were diagnosed by microscopy (Giemsa staining) and nested PCR (32). These studies received ethical clearance from the local ethics committee and the University of So Paulo Ethics Committee.
Recombinant proteins. Glutathione S-transferase (GST) fusion proteins representing the N terminus (ICB2-5; contains 505 amino acids [aa], corresponding to aa 170 to 675 from the original PvMSP1 molecule of the Belem strain [11]) and the C terminus (ICB10; contains 111 amino acids, corresponding to aa 1615 to 1726, and encodes the two epidermal growth factor-like domains of other MSP119 molecules) of PvMSP1 have already been described (19, 34). GST and recombinant proteins were purified on glutathione-Sepharose 4B columns (Amersham Pharmacia), and protein concentration was determined by Bradford assay (Bio-Rad).
Immunoassay (enzyme-linked immunosorbent assay [ELISA]). The presence of naturally acquired antibodies against GST-PvMSP1 tags has been described in detail elsewhere (19, 34). To determine if a serum was positive, we calculated the cutoff for each recombinant protein representing the N terminus and the C terminus of PvMSP1 for each survey, using as negative controls individual sera from nine healthy individuals from Portuchuelo and three healthy individuals from elsewhere who never had a past history of malaria. The cutoff was calculated by the average of ICB2-5 optical density (OD) minus GST OD plus 2 standard deviations (SD). All tests were done in duplicate. Average OD was calculated for each individual, and serum was considered positive if ICB2-5 OD minus GST OD was equal to or greater than the cutoff. The same methodology was applied for ICB10.
IgG subclasses. IgG subclasses were determined by ELISA as described elsewhere (5). Briefly, mouse monoclonal antibodies to each subclass were purchased from Sigma (St. Louis, MO) and diluted according to manufacturer's instructions using a panel of positive sera previously known to react with each subclass. All sera were tested at 1:100 dilutions, and monoclonal antibody binding was detected with peroxidase-conjugated anti-mouse immunoglobulin (Sigma). Positive sera for each isotype were determined as described above for total IgG.
Statistical analyses. Microsoft Access 2.0 (Redmond, WA) was used for database storage. Analysis was performed with SPSS version 10.0 and Epi Info 3.3.2 (Centers for Disease Control and Prevention, Atlanta, GA). Proportions and categorical data were compared by chi-squared test with Yate's correction in cases of 2-by-2 contingency tables or by Fisher's exact test. Odds ratios (OR) and 95% confidence intervals (95% CI) were used to measure associations. Continuous variables analyzed in this data set did not conform to normal distribution and thus were analyzed by Mann-Whitney tests. Multivariate logistic regression analysis was used to evaluate (i) the association between PvMSP1 serology and time of residence in areas were malaria is endemic, controlling for age, and (ii) the association between malaria clinical outcome (symptomatic versus asymptomatic infection) and serology against the PvMSP1 N terminus and C terminus in survey A, controlling for age. Kaplan-Meier survival analysis was performed to compare the probability of P. vivax infection over the 1-year follow-up period (September 1998 to September 1999) in the groups of individuals with positive and negative serology against PvMSP1 N and C termini in survey A. Because malaria is seasonal in Portuchuelo (Fig. 1), only individuals who remained in Portuchuelo the entire study period were included in the survival analysis. Moreover, individuals who were found infected in survey A were excluded due to the uncertainty of their serology against PvMSP1 at the time they were infected. Because individuals living in Sector B (Fig. S1) of Portuchuelo are at higher risk of malaria infection (17), a Cox proportional hazard model was further used to evaluate the risk of P. vivax infection by serology, controlling for geographical sector.
RESULTS
Prevalence of antibodies against PvMSP1 in the study area. The prevalence of anti-PvMSP1 antibodies was determined using immune sera collected from individuals living in Portuchuelo in three different cross-sectional surveys: survey A, September 1998 (n = 173); survey B, March 1999 (n = 125); and survey C, September 1999 (n = 131). Moreover, as both the N and C termini of PvMSP1 are immunogenic in natural infections (19, 34), we determined the prevalence of anti-PvMSP1 antibodies against each of these regions using GST-recombinant proteins representing the N terminus and the C terminus in ELISA assays. It is notable that during the year of the study, the incidence of P. vivax infections per 1,000 inhabitants was seasonal, peaking at the end of the rainy season when the second survey (B) was performed (Fig. 1). In spite of these marked differences in incidences during the study year, the prevalence of anti-PvMSP1 antibodies against the N and C termini of PvMSP1 was not significantly different in all three surveys (N terminus for survey A, 36.4%; N terminus for survey B, 32.8%; N terminus for survey C, 35.1%; C terminus for survey A, 42.8%; C terminus for survey B, 42.4%; C terminus for survey C, 38.9%). Moreover, although the overall prevalence of antibodies against the PvMSP1 C terminus in each survey was higher than that of antibodies against the PvMSP1 N terminus, these differences were not statistically significant (A, P = 0.23; B, P = 0.12; and C, P = 0.52). It was also observed that the presence of antibodies to the N terminus was associated with the presence of antibodies to the C terminus of PvMSP1 in surveys A and B (P = 0.002 for both), while in survey C, although the same trend was observed, the difference was not statistically significant (P = 0.085). These results showed that the N and C termini of PvMSP1 are immunogenic in natural infections among the residents of Portuchuelo and validated their use as markers for looking for associations with reduced risk of infection and clinical protection.
Dependency of antibody responses to PvMSP1 and time of residence in areas where malaria is endemic, age, and time since last malaria attack. To determine whether the antibody responses against PvMSP1 were dependent on the time of residence in areas where malaria is endemic, we grouped time of residence as 0 to 7, 8 to 14, 15 to 29, and more than 30 years based on 25th percentile of all three cross-sectional surveys (Tables 1 and 2). A multiple logistic regression analysis was performed to evaluate if serology against PvMSP1 was associated with time living in areas where malaria is endemic, controlling for age. Odds ratios (with 95% CI) were calculated for each group, using 0 to 7 years as the reference group. The results demonstrated that individuals living longer in areas where malaria is endemic had increasingly higher chances of having naturally acquired IgG antibodies against the N terminus but not against the C terminus of PvMSP1. Thus, for the N terminus there was no statistically significant difference between the reference group and the group of 8 to 14 years. However, individuals living in areas where malaria is endemic for 15 to 29 years or for 30 or more years had 30 times greater chances of having acquired antibodies against the PvMSP1 N terminus (Table 1). Similar results were observed in surveys B and C, although for survey C it was not possible to run the logistic regression and calculate the odds ratio, because no individuals in the reference group had positive serology (but the same trend was clear; 2 = 67.5, P < 0.001). In contrast, the same analysis for the C terminus of PvMSP1 showed no statistically significant differences among the groups in the three surveys (Table 2). These results demonstrate that acquisition of antibodies to the N terminus but not the C terminus of PvMSP1 in residents of Portuchuelo depends on the length of residence in areas where malaria is endemic.
We next analyzed if there was a dependency in the acquisition of antibodies against the N and C termini of PvMSP1 with age and the amount of time since the last malaria attack. As these parameters did not present a normal distribution for this population, we used the nonparametric test of Mann-Whitney for analysis. As expected, the results with regard to age demonstrated that individuals with positive serology against the N terminus of PvMSP1 are older than individuals with negative serology for all three surveys (survey A, median = 38 versus 13 years, Mann-Whitney = 1,468, P < 0.0001; survey B, median = 25 versus 13 years, Mann-Whitney = 955.5, P < 0.0001; survey C, median = 37 versus 11 years, Mann-Whitney = 582.5, P < 0.0001). Similar results were observed for the PvMSP1 C terminus only in survey A (median = 20.5 versus 16 years, Mann-Whitney = 2,981, P = 0.036). For surveys B and C, the difference was not significant (survey B, median = 21 versus 15.5 years, Mann-Whitney = 1,540, P = 0.07; survey C, median = 15 versus 13 years, Mann-Whitney = 1,617, P = 0.06). This result is likely due to the fact that the risk of infection is the same for all age groups in Portuchuelo (1), and there is a natural boost against the PvMSP1 C terminus but not the N terminus (34). Thus, upon infection during survey B, many different individuals reacted and remained positive against the PvMSP1 C terminus independently of age.
Analysis of the dependency of anti-PvMSP1 antibodies on the amount of time since the last malaria attack revealed that individuals with positive serology against the PvMSP1 N terminus had their most recent malaria attack significantly farther in the past than those with negative serology (survey A, median = 6 versus 2 years, Mann-Whitney = 1,449, P < 0.0001). In contrast, there were no significant differences for individuals with positive and negative serology against the PvMSP1 C terminus and the amount of time since the last malaria attack (survey A, median = 2 versus 3 years, Mann-Whitney = 2,698, P = 0.61). Similar results for both PvMSP1 termini were observed in surveys B and C (data not shown). Together, these results showed a dependency on the acquisition of IgG antibodies against the N terminus of PvMSP1 and time of residence in areas where malaria is endemic, age, and time since the last malaria attack. The same dependency was not observed for the C terminus of PvMSP1. As time of residence in areas where malaria is endemic had been associated with acquired immunity in the epidemiological study of the same human population (1), we next determined if an association could be established with PvMSP1, risk of infection, and clinical protection.
Association of antibody responses to PvMSP1 with risk of P. vivax infection and clinical protection. To compare the probability of P. vivax infection in the groups of individuals with positive and negative responses against PvMSP1 in survey A, a survival analysis of time to P. vivax infection over the 1-year follow-up period was performed. The Kaplan-Meier cumulative risk of P. vivax infection at the end of the study year was significantly lower for individuals with positive serology in survey A than for individuals with negative serology against the PvMSP1 N terminus (11.9% versus 31.8%, respectively; P = 0.0186). In contrast, this association was not significant for individuals with positive or negative serology against the PvMSP1 C terminus (26.9% versus 23.5%, respectively; P = 0.7414) (Fig. 2). Because individuals living in Sector B of Portuchuelo are at higher risk of malaria infection (17), a Cox proportional hazard model was further used to evaluate the relative risk of P. vivax infection for the groups of individuals with positive and negative serology against PvMSP1, controlling for geographical sector. Indeed, the risk of P. vivax infection after adjusting for sector was three times higher for individuals with negative serology than for individuals with positive serology against the N terminus (hazard ratio, 2.954; 95% CI, 1.140 to 7.652; P = 0.026) but not the C terminus (hazard ratio, 0.892; 95% CI, 0.447 to 1.778; P = 0.745) of PvMSP1.
Having established that individuals with positive serology against the PvMSP1 N terminus had reduced risk of P. vivax infection, we next performed a multivariate logistic regression analysis to determine if malaria clinical outcome (symptomatic versus asymptomatic) was associated with positive serology against PvMSP1 in survey A, controlling for age. As shown in Table 3, individuals with positive serology against the N terminus had chances of having asymptomatic P. vivax infection, after adjusting for age, that were 12 times greater than those of individuals with positive serology against the PvMSP1 C terminus. Together, these data show that individuals with positive serology against the PvMSP1 N terminus but not the C terminus are protected against P. vivax infection, and when they are infected, they are likely to be symptomless.
IgG subclass response to PvMSP1 N and C termini. To determine if there was a particular IgG subclass correlated with protection, we examined the IgG isotype responses of patients presented in the three surveys who had a demonstrated presence of PvMSP1 total IgG. No IgG2 or IgG4 response to either the N or C terminus of PvMSP1 was detected (data not shown). In contrast, there were marked differences in the frequencies of IgG1 and IgG3 isotype responses in all three surveys to the N and C termini (Fig. 3). Thus, IgG1 isotype response was of low frequency or was nondetectable in all three surveys for either region of PvMSP1 (Fig. 3). In striking contrast, high frequencies of IgG3 isotype response were detected to the N terminus (56% in survey A, 63.2% in survey B, and 69.6% in survey C) but not to the C terminus (0% in survey A, 16% in survey B, and 3.6% in survey C) of PvMSP1. Indeed, the frequency of IgG3 response was significantly higher for the N terminus than the C terminus for all three surveys (P < 0.003).
Longitudinal analysis of anti-PvMSP1 antibodies in asymptomatic versus symptomatic P. vivax individuals from Portuchuelo. We next examined the relative levels of anti-PvMSP1 antibodies in asymptomatic and symptomatic P. vivax individuals from Portuchuelo in the three cross-sectional surveys. To do so, we recorded individual ELISA OD minus GST OD values of the sera against the recombinant proteins representing the N terminus and C terminus of PvMSP1. Responses were classified as negative (OD < cutoff) or positive; positives were subgrouped according to the 33rd and 66th percentiles as low (cutoff < OD < 33rd percentile), middle (33rd percentile < OD < 66th percentile), and high (OD p66th percentile). As shown in Fig. S2 in the supplemental material, asymptomatic individuals present a higher proportion and magnitude of response to the N terminus of PvMSP1 than do symptomatic individuals, while this difference is not observed for the C terminus of PvMSP1.
DISCUSSION
In this work, a prospective 1-year longitudinal study of a human population in the Brazilian Amazon Basin in which asymptomatic P. vivax patients had been previously identified was performed to determine if the presence of naturally acquired antibodies against the N terminus and/or C terminus of the merozoite surface protein 1 of Plasmodium vivax (PvMSP1) was associated with reduced risk of P. vivax infection and clinical protection. The results demonstrated that close to 33% and 40% of the population contained anti-PvMSP1 antibodies against the N and C termini of PvMSP1, respectively, independent of seasonal transmission rates. Moreover, the data showed that individuals living in areas where malaria is endemic for 15 to 29 years or 30 years had 30 times greater chances of having acquired antibodies against the PvMSP1 N terminus, but not the C terminus, than individuals living <7 years in areas where malaria is endemic. Acquisition of antibodies against the N terminus but not the C terminus of PvMSP1 was dependent on age and the amount of time since the last malaria attack. Moreover, when an association of anti-PvMSP1 antibodies with reduced risk of P. vivax infection and clinical protection was investigated in the individuals who participated in this study for the entire year, we found that this association could only be established with antibodies against the N terminus but not the C terminus of PvMSP1 and that most asymptomatic individuals presented IgG antibodies predominantly of the immunoglobulin G3 subclass against the N terminus of PvMSP1.
The existence of asymptomatic plasmodial infections in Brazil allowed the initiation of studies to look for associations of antibodies against parasite antigens with clinical protection (5, 23). Interestingly, these two reports used the same human populations to look for associations of acquired immunity with IgG subclasses against the C terminus of the MSP1 proteins from P. falciparum (5) and P. vivax (23). The criteria used to define asymptomatic individuals in these studies (Giemsa-positive or PCR-positive patients that did not develop symptoms after 72 h of this detection), however, is not stringent enough to discount the possibility that these could later turn into symptomatic patients. Regardless, the results suggested that there is an association of clinical protection with IgG1 subclass antibodies against the C terminus of P. falciparum (5), whereas no association with regard to antibodies or subclasses could be supported against the C terminus of P. vivax (23).
In contrast to the above studies, we looked for associations of clinical protection and reduced risk of infection with serology against PvMSP1 in a human population in which symptomless P. vivax-infected individuals have been unarguably found. In fact, one of us (F.A.P.) personally diagnosed these patients, and after obtaining ethical consent to not treat them with drugs, actively followed them up twice a week for 2 months to guarantee that they did not develop malaria symptoms. Moreover, we analyzed independently the N and C termini of PvMSP1, as antibodies against both regions of the MSP1 of P. falciparum have been associated with clinical protection and acquired immunity (8, 10, 12, 14, 24, 25, 26). Strikingly, in spite of being the most immunogenic portion of PvMSP1 in natural infections and being capable of boosting (34), there was no association of antibody responses against the C terminus of PvMSP1 in survey A with clinical protection or risk of P. vivax infection (Fig. 2 and Table 3). This result is thus in agreement with the one reported previously for this portion of PvMSP1 (23). In contrast, immune sera from these same patients revealed a highly statistically significant association of antibodies against the N terminus of PvMSP1 with clinical protection and reduced risk of P. vivax infection over the 1-year follow-up period. Prospective studies with other human populations in which symptomless P. vivax-infected individuals are solidly identified should validate whether this finding is a particularity of individuals from Portuchuelo or a general finding for vivax malaria in Brazil and elsewhere.
It is now amply recognized that clinical protection against the asexual blood stages in falciparum malaria is associated with the cytophilic IgG1 and IgG3 subclass antibodies directed against merozoite surface antigens (3, 4). Indeed, solid associations of clinical protection of IgG3 isotype antibodies against MSP2 in the Solomon Islands and in The Gambia (29, 37) and of the IgG1 isotype to RAP1 in reference 15 have been reported. In the particular case of the MSP1 protein of P. falciparum, initial studies using recombinant proteins representing the N and C termini of PfMSP1 clearly demonstrated that there is a marked dichotomy in the IgG subclass response to these regions (7). Thus, the IgG3 subclass response is predominantly against block 2 from the N terminus, whereas the IgG1 subclass response is predominantly against MSP119 at the C terminus. Our findings demonstrated that only the IgG3 subclass response is frequently detected in asymptomatic individuals, and this response is against the N terminus but not the C terminus of PvMSP1. Further work needs to be done to determine why merozoite surface antigens are able to elicit such strikingly different IgG subclass responses to different regions of the same molecules and the consequences of such responses with regard to clinical aspects of malaria.
An interesting observation of our results is that high sustained levels of antibodies against the N terminus of PvMSP1 were only detected in asymptomatic individuals, whereas in symptomatic patients, most of them did not recognize this recombinant protein (Fig S2). In comparison, similar levels of antibodies against the C terminus of PvMSP1 were seen in both asymptomatic and symptomatic patients. Sustained high antibody levels against the N terminus of PvMSP1 in asymptomatic patients can be explained by the presence of circulating, mostly undetectable parasites, which should facilitate the formation and migration of specific plasma cells to the bone marrow (21). Lack of high levels of antibodies against the N terminus of PvMSP1 in symptomatic patients is far more difficult to explain, but it seems clear that an active mechanism precludes most of these patients from developing an antibody response against this region of PvMSP1. Whether this observation and mechanism are indeed related to acquired immunity in P. vivax remains to be determined.
The existence of asymptomatic P. vivax-infected individuals poses a major problem for eradication control strategies in regions where malaria is endemic, as they represent reservoirs that maintain circulating parasites. Giemsa staining and PCR first helped in the identification of such asymptomatic carriers. Our studies point to the potential value of the PvMSP1 antibodies, in particular of the IgG3 subclass against the N terminus of PvMSP1, as seroepidemiological markers that, together with information on time of residence in areas where malaria is endemic, can indicate the existence of clinically protected individuals in a population. Active surveillance of human populations using these three techniques (microscopy, PCR, and serology) together with epidemiological data should unveil most asymptomatic carriers and guide local health policies to treat them with drugs as an additional malaria control strategy. It has been shown that such a procedure significantly reduces malaria cases (F. P. Alves, L. H. Pereira da Silva, and E. P. Camargo, unpublished data).
In summary, our study is the first to demonstrate an association of clinical protection and reduced risk of infection with naturally acquired IgG antibodies, predominantly of the IgG3 subclass, against a P. vivax antigen, PvMSP1. This association was with the N terminus but not the C terminus of PvMSP1, in spite of the latter being the most immunogenic portion of the molecule and the only portion capable of boosting in natural infections. It is important to emphasize, however, that this association does not imply that antibody responses to the PvMSP1 N terminus is by itself the mechanism of protection or simply a marker of it. Regardless, fine epitope mapping of N-terminus PvMSP1 sequences associated with this clinical protection and reduced risk of infection should guide rational approaches to develop a subunit PvMSP1 N-terminus vaccine.
ACKNOWLEDGMENTS
This work was supported by the Fundao de Amparo a Pesquisa do Estado de So Paulo (FAPESP), Coordenao de Aperfeioamento de Pessoal de Nível Superior (CAPES), Programa de Apoio a Núcleos de Excelência (PRONEX) do Ministerio da Ciência e Tecnologia, Brasil, and Program PAL+ (Recherche sur le paludisme et les maladies transmissibles associees pour les pays em development), Institut de Recherche pour le Development, France. The laboratory of H.A.P. receives support from FAPESP (01/09401-0) and CNPq (302572/2002-3).
We are particularly grateful to Irene Soares for titrations and helpful discussions on malarial MSP1 IgG subclasses and to Andrea Sendoda and Ricardo Raele for helping in purifying recombinant proteins and aliquoting the sera in 96-well plates.
Supplemental material for this article may be found at http://iai.asm.org/.
These authors contributed equally to this work.
Present address: DKFZ INF 280, 69120 Heidelberg, Germany.
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